This project focuses on the functions of protein kinases and protein phosphatases in transducing extracellular signals initiated by hormones, neurotransmitters, growth factors, and antigens. Covalent modification of proteins by phosphorylation/dephosphorylation plays a central role in mediating many cellular events resulting from ligand-receptor interaction and generation of a variety of second messengers. Several protein kinases, especially the Ca2+/phospholipid/diacylglycerol- stimulated protein kinase C family, and phosphatases have been prepared from rat brain for the identification of protein substrates modified by these enzymes. Several novel proteins have been isolated by ion-exchange and reverse phase chromatography and their partial amino acid sequences determined. The effects of phospholipids and Ca2+-binding proteins, calmodulin, and S-100 protein on the phosphorylation/dephosphorylation of these proteins are being investigated. In addition, modification of the protein kinase C substrate, neurogranin, by oxidizing agents such as hydrogen peroxide and nitric oxide has been shown to influence the kinase-catalyzed phosphorylation. Molecular cloning of the cDNAs of these novel protein kinase C substrates is in progress. The genomic structures of the CNS-specific protein kinase ~ isozyme and its substrate, neurogranin, have been elucidated. The mechanisms controlling the tissue-specific and developmental stage-regulated expression of these two genes are currently being investigated.